Vías de degradación de las proteínas α-sinucleína y DJ-1 / PARK7 implicadas en la patogénesis de la enfermedad de Parkinson

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 13-09-2019Esta tesis tiene embargado el acceso al texto completo hasta el 13-03-2021La degradación de proteínas juega un papel fundamental en la proteostasis celular siendo uno de los factores más relevantes implicados en el desarrollo de las enfermedades neurodegenerativas. Las dos vías mayoritarias de degradación de proteínas intracelulares son la vía de la ubicuitina proteasoma y la vía autofágica lisosomal. Este trabajo aborda el estudio de las rutas proteolíticas que participan en la degradación de α-sinucleína y DJ-1, proteínas implicadas en la patogénesis de la enfermedad de Parkinson. Dentro de la vía autofágica lisosomal, se ha descrito que la autofagia mediada por chaperonas está implicada en la degradación de α-sinucleína, DJ-1 y otros sustratos proteicos como Iκβα, Rcan1 y Gapdh. Lamp-2A es la isoforma de Lamp-2 considerada como el receptor de membrana lisosomal para la degradación de proteínas por la vía de la autofagia mediada por chaperonas. En esta tesis se muestra que la interrupción o el silenciamiento de la expresión de Lamp-2 no resulta en un incremento de los niveles en estado estacionario de las proteínas mencionadas anteriormente, como cabría esperar, y tampoco se encuentran diferencias significativas en su degradación entre células control y deficientes en Lamp-2. Por otro lado, DJ-1 silvestre es una proteína con una vida media larga y sus niveles no se alteran por el tratamiento de las células durante 24 horas con inhibidores de la función lisosomal, de la síntesis de proteínas o por la activación de la vía autofágica lisosomal por ayuno de suero. El estudio sistemático de la degradación de DJ-1 silvestre y los mutantes de cambio de sentido sin etiquetar transfectados tanto en fibroblastos embrionarios procedentes de ratones deficientes en DJ-1 (mimetizando el contexto celular de los pacientes de enfermedad de Parkinson portadores de mutaciones homocigóticas) como en células expresando DJ-1 endógeno, muestra que los mutantes puntuales DJ-1 A39S, E64D, R98Q, A104T, D149A, K175E y A179T tienen una vida media larga, similar a la de DJ-1 silvestre y que los mutantes DJ-1 L10P, M26I, A107P, P158Δ, E163K, L166P y L172Q son proteínas inestables. La utilización de inhibidores del proteasoma o de la función lisosomal no es efectiva para prevenir la degradación de estos mutantes inestables de DJ-1. El análisis de la localización subcelular por estudios de inmunofluorescencia indirecta y de fraccionamiento subcelular muestra que los mutantes inestables A107P, P158Δ, E163K, L166P y L172Q se asocian claramente con la mitocondria donde son degradados por la proteasa LonP1 de la matriz mitocondrial, lo que pone de manifiesto, por primera vez, la existencia de una nueva vía de degradación mitocondrial para estos mutantes de DJ-1 patogénicos en la enfermedad de ParkinsonProtein degradation plays a major role in cellular proteostasis, being one of the main factors involved in the development of neurodegenerative diseases. The two main protein degradation pathways in cells are the ubiquitin proteasome system and the autophagic lysosomal system. This work tackles the study of the proteolytic pathways implicated in the degradation of α-synuclein and DJ-1, proteins associated with the pathogenesis of Parkinson disease. It has been reported that chaperone-mediated autophagy is implicated in the degradation of α-synuclein, DJ-1 and other proteins including Iκβα, Rcan1 and Gapdh. Lamp-2A isoform is considered the lysosomal receptor for the uptake of proteins being degraded by the chaperone-mediated pathway. In this thesis, it is shown that interruption or silencing of Lamp-2 expression does not result in an increase in the steady-state levels of the previously mentioned proteins, as would be expected. Furthermore, there are no significant changes in the degradation rates of these proteins between control and Lamp-2-deficient cells. On the other hand, DJ-1 wild type has a long half-life and its protein levels remain unchanged after treatment of cells for 24 hours with inhibitors of lysosomal function and protein synthesis or after activation of the autophagic lysosomal pathway by serum starvation. The systematic study of the degradation of untagged DJ-1 wild type and missense mutants transfected both in mouse embryonic fibroblasts obtained from DJ-1 null mice (mimicking the situation of cells from patients carrying homozygous mutations) and cells expressing endogenous DJ-1, shows that the DJ-1 missense mutants A39S, E64D, R98Q, A104T, D149A, A171S, K175E and A179T are as stable as DJ-1 wild type, whereas the DJ-1 missense mutants L10P, M26I, A107P, P158Δ, E163K, L166P and L172Q are unstable proteins. Inhibition of the proteasome or the lysosomal function is ineffective for prevention of the degradation of these unstable DJ-1 missense mutants. Subcellular localization studies by indirect immunofluorescence and biochemical fractionation show that M26I, A107P, P158Δ, E163K, L166P and L172Q missense mutants are clearly associated with mitochondria where are degraded by the matrix protease LonP1. These results reveal, for the first time, the existence of a new degradation pathway in the mitochondria of these pathogenic mutants associated with Parkinson diseas

Proteins Directly Interacting with Mammalian 20S Proteasomal Subunits and Ubiquitin-Independent Proteasomal Degradation

The mammalian 20S proteasome is a heterodimeric cylindrical complex (α7β7β7α7), composed of four rings each composed of seven different α or β subunits with broad proteolytic activity. We review the mammalian proteins shown to directly interact with specific 20S proteasomal subunits and those subjected to ubiquitin-independent proteasomal degradation (UIPD). The published reports of proteins that interact with specific proteasomal subunits, and others found on interactome databases and those that are degraded by a UIPD mechanism, overlap by only a few protein members. Therefore, systematic studies of the specificity of the interactions, the elucidation of the protein regions implicated in the interactions (that may or may not be followed by degradation) and competition experiments between proteins known to interact with the same proteasomal subunit, are needed. Those studies should provide a coherent picture of the molecular mechanisms governing the interactions of cellular proteins with proteasomal subunits, and their relevance to cell proteostasis and cell functioning

Correction: Inhibitors of lysosomal function or serum starvation in control or LAMP2 deficient cells do not modify the cellular levels of Parkinson disease-associated DJ-1/PARK 7 protein.

[This corrects the article DOI: 10.1371/journal.pone.0201152.]

Lysine-Less Variants of Spinal Muscular Atrophy SMN and SMNΔ7 Proteins Are Degraded by the Proteasome Pathway

Spinal muscular atrophy is due to mutations affecting the SMN1 gene coding for the full-length protein (survival motor neuron; SMN) and the SMN2 gene that preferentially generates an exon 7-deleted protein (SMNΔ7) by alternative splicing. To study SMN and SMNΔ7 degradation in the cell, we have used tagged versions at the N- (Flag) or C-terminus (V5) of both proteins. Transfection of those constructs into HeLa cells and treatment with cycloheximide showed that those protein constructs were degraded. Proteasomal degradation usually requires prior lysine ubiquitylation. Surprisingly, lysine-less variants of both proteins tagged either at N- (Flag) or C-terminus (V5) were also degraded. The degradation of the endogenous SMN protein, and the protein constructs mentioned above, was mediated by the proteasome, as it was blocked by lactacystin, a specific and irreversible proteasomal inhibitor. The results obtained allowed us to conclude that SMN and SMNΔ7 proteasomal degradation did not absolutely require internal ubiquitylation nor N-terminal ubiquitylation (prevented by N-terminal tagging). While the above conclusions are firmly supported by the experimental data presented, we discuss and justify the need of deep proteomic techniques for the study of SMN complex components (orphan and bound) turn-over to understand the physiological relevant mechanisms of degradation of SMN and SMNΔ7 in the cell

Protein expression levels of DJ-1 following activation of CMA by serum starvation in control and Lamp-2-deficient N2a cells.

<p>Exponentially growing control and Lamp-2-deficient N2a cells were kept in complete medium (C) or starved of serum for 24 h in the absence (St) or in the presence of NH₄Cl, NH₄Cl and leupeptin (leup), or 3-methyl adenine (3-MA). Panels A and B show the effect of serum starvation in N2a shRNA scrmbl cells and in Lamp-2-deficient N2a shRNA Lamp-2 cells, respectively. Total cell lysates were analysed by Western and immunoblot with the corresponding specific antibodies. Anti-tubulin antibodies were used as total protein loading control. Graphs below each panel show the quantification of the levels of the different proteins analysed respect to the levels in cells kept in complete growth medium, controls. Values are expressed as mean ± s.e.m. from three different experiments. Significant differences between groups ** at p<0.01 by Student t-test are indicated.</p

Protein expression levels of DJ-1 following activation of autophagy by serum starvation in control and Lamp-2-deficient MEF cells.

<p>Exponentially growing control and Lamp-2-deficient MEF cells were kept in complete medium (C) or starved of serum for 24 h in the absence (St) or in the presence of NH₄Cl, NH₄Cl and leupeptin (leup), or 3-methyl adenine (3-MA). Panels A and B show the effect of serum starvation in MEF wild type (MEF Wt) and Lamp-2-deficient MEF (Lamp-2^-/y) cells, respectively. Total cell lysates were analysed by Western and immunoblot with the corresponding specific antibodies, as indicated. Anti-tubulin antibodies were used as total protein loading control. Graphs below each panel show the quantification of the levels of the different proteins analysed respect to the levels in cells kept in complete growth medium, controls. Values are expressed as mean ± s.e.m. from three different experiments. Significant differences between groups ** at p<0.01 by Student t-test are indicated.</p

Stability of DJ-1 protein in control and Lamp-2-deficient cell lines after inhibition of protein synthesis.

<p>Exponentially growing cells from control and Lamp-2-deficient cells were treated with cycloheximide (CHX) for the times indicated. Total cell lysates were prepared and DJ-1 protein levels were analysed by Western and immunoblot with specific antibodies. Anti-tubulin antibodies were used as total protein loading control. Panels show the results obtained for MEF (A), N2a (B) and B-LCLs (C), Graphs on the right show the quantification of the levels of DJ-1 protein respect to untreated cells as controls (time 0 h). Values are expressed as mean ± s.e.m. from three different experiments. n.s. not significant difference.</p

Protein expression levels of DJ-1 in control and Lamp-2-deficient cell lines in the presence of inhibitors of lysosomal function.

<p>Exponentially growing cells from control and Lamp-2-deficient cells were incubated in complete medium r suplemented with 20mM NH₄Cl or 20 mM NH₄Cl in combination with 50 μM leupeptin (leup) for 24 h. Total cell lysates were analysed by Western and immunoblot with the corresponding specific antibodies: DJ-1 and LC-3. Anti-tubulin antibodies were used as total protein loading control. Panels show the results for MEF (A), N2a (B), B-LCLs (C) and SN4741 (D). Graphs below each panel show the quantification of the levels of the proteins analysed respect to the levels in cells kept in complete growth medium, controls. Values are expressed as mean ± s.e.m. from three different experiments. Significant differences between groups ** at p<0.01 by Student t-test are indicated.</p

Protein expression levels of DJ-1 following activation of CMA by serum starvation in SN4741 cells.

<p>Exponentially growing SN4741 cells were kept in complete medium (C) or starved of serum for 24 h in the absence (St) or in the presence of NH₄Cl, NH₄Cl and leupeptin (Leup), or 3-methyl adenine (3-MA). Panels show the effect of serum deprivation in SN4741. Total cell lysates were analysed by Western and immunoblot with the corresponding specific antibodies, as indicated. Anti-tubulin antibodies were used as total protein loading control. Graphs below each panel show the quantification of the levels of the different proteins analysed respect to the levels in cells kept in complete growth medium, controls. Values are expressed as mean ± s.e.m. from three different experiments. Significant differences between groups ** at p<0.01 by Student t-test are indicated.</p

Protein expression levels of DJ-1 following activation of CMA by serum starvation in control and Lamp-2-deficient B-LCLs.

<p>Exponentially growing control and Lamp-2-deficient B-LCL were kept in complete medium (C) or starved of serum (8h) in the absence (St) or in the presence of NH₄Cl, NH₄Cl and leupeptin (Leup), or 3-methyl adenine (3-MA). Panels A and B show the effect of serum starvatrion in B-LCLs control and Lamp-2 (-). Lamp-2-deficient B-LCL, respectively. Total cell lysates were analysed by Western and immunoblot with the corresponding specific antibodies, as indicated. Anti-tubulin antibodies were used as total protein loading control. Graphs below each panel show the quantification of the levels of the different proteins analysed respect to the levels in cells kept in complete growth medium, controls. Values are expressed as mean ± s.e.m. from three different experiments. Significant differences between groups ** at p<0.01 by Student t-test are indicated.</p